Transgenic plant cells and plants having modified activity of the gbssi and of the be protein

ABSTRACT

Transgenic plant cells and plants are described which synthesize a starch which is modified in comparison to wild-type plant cells and plants and show a decrease in the activity of GBSSI and BE proteins. Furthermore, the modified starches obtainable from these plant cells and plants are described, and processes for their preparation.

The present invention relates to transgenic plant cells and plantshaving a decreased activity of a GBSSI protein and a decreased activityof a BE protein, in particular of a BEI protein, and to means andprocesses for their production. Plant cells and plants of this type cansynthesize a modified starch which has an amylopectin content of atleast 90% and in comparison to starch from corresponding plants of thewaxy phenotype an increased phosphate content and/or a decreasedgelatinization temperature. The present invention therefore also relatesto the starch synthesized from the plant cells and plants according tothe invention and to processes for the production of this starch. Thepresent invention further relates to the use of certain nucleic addmolecules for the production of plants which synthesize a starch havingan amylopectin content of at least 90%, which in comparison to starchfrom corresponding plants of the waxy phenotype has an increasedphosphate content and/or a decreased gelatinization temperature.

In view of the increasing importance which has recently been attributedto plant constituents as renewable raw material sources, it is one ofthe objects of biotechnological research to make an effort to adaptthese plant raw materials to the requirements of the processingindustry. In order to make possible application of renewable rawmaterials in as many areas of use as possible, it is moreover necessaryto achieve a wide variety of substances.

In addition to oils, fats and proteins, polysaccharides are essentialrenewable raw materials from plants. In addition to cellulose, a centralposition in the case of the polysaccharides is occupied by starch, whichis one of the most important storage substances in higher plants. Cornis one of the most interesting plants here, as it is the most importantcrop plant globally for starch production.

The polysaccharide starch is a polymer made from chemically uniformmonomers, the glucose molecules. However, it is a very complex mixtureof different forms of molecules which differ with respect to theirdegree of polymerization and the occurrence of branching of the glucosechains. Starch is therefore not a uniform raw material. Adifferentiation is made in particular between amylose starch, anessentially unbranched polymer of α-1,4-glycosidically linked glucosemolecules, and amylopectin starch, which for its part is a complexmixture of differently branched glucose chains. The branchings comeabout owing to the occurrence of additional α-1,6-glycosidic links. Intypical plants used for starch production, such as, for example, corn orpotatoes, the synthesized starch consists to about 20%-30% of amylosestarch and to about 70%-80% of amylopectin starch.

In order to make possible the widest possible use of starch, it appearsdesirable to make available plants which are able to synthesize modifiedstarch which is particularly suitable fir various intended uses. Onepossibility of making available plants of this type consists—in additionto breeding measures—in the controlled genetic modification of thestarch metabolism of starch-producing plants by genetic engineeringmethods.

The ratio of amylopectin to amylose has a great influence on thephysico-hemical properties of the starches and thus on the particularapplication possibilities of these starches. Since processes for theseparation of these two components are very time consuming andcost-intensive, processes of this type are no longer used on a largeindustrial scale (Young, A. H. in: Starch Chemistry and Technology. Eds.R. L. Whistler, J. N. BeMiller and E. F. Paschall. Academic Press, NewYork, 1984, 249-283). For a large number of applications, it would thusbe desirable to have available starches which only contain one of thetwo polymers.

Hitherto, both mutants and plants produced by genetic engineeringprocesses have been described which have a modified amylopectin/amyloseratio in comparison to corresponding wild-type plants. For example, aso-called “waxy” mutant from corn, which has a mutation in the genecoding for the granule-bound starch synthase 1, abbreviated: GBSSI(Akasuka and Nelson, J. Biol. Chem., 241, (1966), 2280-2285; Shure etal., Cell 35 (1983), 225-233), produces a starch which essentiallyconsists of amylopectin. Below, a waxy starch is understood as meaning astarch having an amylopectin content of at least 90%.

For potatoes, genotypes were produced both by chemical mutagenesis of ahaploid line (Hovenkamp-Hermelink et al., Theor. Appl. Genet., 225,(1987), 217-221) and by antisense inhibition of the gene for the GBSSI,whose starches essentially consist of amylopectin starch. In comparisonto starches from corresponding wild-type plants, waxy potato starches ofthis type have no differences, with respect to the phosphate content, inthe morphology of the starch granule or in the ion content (Visser etal., Starch/Stärke, 49, (1997), 438-443).

In addition to the amylose/amylopectin ratio, the functional propertiesof the starch are strongly influenced by the phosphate content, themolecular weight, the pattern of side chain distribution, the content ofions, the lipid and protein content etc. Examples of importantfunctional properties which can be mentioned here are the solubility,the retrogradation behavior, the water-binding ability, the film-formingproperties, the viscosity, the gelatinization properties, the stabilityetc. The size of the starch granule can also be of importance forvarious applications.

The phosphate content can basically be modified both by geneticengineering approaches (see, for example, WO 97/11188-A1; Safford etal., Carbohydrate Polymers 35, (1998), 155-168) and by subsequentchemical phosphorylation (see, for example, in: Starch Chemistry andTechnology. Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall.Academic Press, New York, 1988, 349-364). However, as a rule chemicalmodifications are cost- and time-intensive.

Until now, it has not been possible to produce plant cells and plantswhich synthesize waxy starches having an increased phosphate contentand/or decreased gelatinization temperature in comparison to starchesfrom corresponding plant(s) (cells) of the waxy phenotype. Proceduresfor the production of plant cells and plants of this type and proceduresfor the production of starches of this type are as yet not described inthe prior art.

Since the phosphate content of the starches influences their properties,it would be desirable to make available plant cells and plants whichsynthesize waxy starches having modified structural and/or functionalproperties in comparison to corresponding plant cells and plants of thewaxy phenotype.

The present invention is thus based on the object of making availableplant cells and plants which, in comparison to corresponding plant cellsand plants of the waxy phenotype, synthesize starches having modifiedstructural and/or functional properties, and waxy starch which differsin its structural and/or functional properties from other waxy starchesand is thus better suited for general and/or specific intended uses.

This object is achieved by the provision of the embodiments described inthe patent claims.

The present invention thus relates to transgenic plant cells which aregenetically modified, the genetic modification leading to a decrease inthe activity of one or more GBSSI proteins occurring endogenously in theplant cell and to a decrease in the activity of one or more BE proteinsoccurring endogenously in the plant cell, in comparison to correspondingnon genetically modified plant cells of wild-type plants.

The genetic modification can be any genetic modification which leads toa decrease in the activity of an endogenous GBSSI protein and of a BEprotein occurring in the plant cell in comparison to non geneticallymodified plant cells of corresponding wild-type plants.

The term “transgenic” in this connection means that the plant cellsaccording to the invention differ in their genetic information fromcorresponding non genetically modified plant cells on account of agenetic modification, in particular the introduction of one or moreforeign nucleic acid molecules.

The term “genetically modified” in this connection means that thegenetic information of the plant cell is modified by introduction of oneor more foreign nucleic acid molecules and that the presence or theexpression of the foreign nucleic acid molecule leads to a phenotypicmodification. “Phenotypic modification” preferably means a measurablemodification in one or more functions of the cells; plant cellsaccording to the invention in particular show a decrease in theexpression of at least one endogenous GBSSI gene and at least oneendogenous BE gene and/or a decrease in the activity of at least oneGBSSI protein and at least one BE protein.

The term “GBSSI protein” is understood in the context of the presentinvention as meaning any protein which in contrast to the class ofsoluble starch synthases belongs to the class of granule-bound starchsynthases isoform I (=GBSSI, EC 2.4.1.21). Plants in which the enzymeactivity of this protein is greatly or completely reduced synthesize anessentially amylose-free, so-called waxy starch (Shure et al., (1983)supra; Hovenkamp-Hermelink et al., (1987), supra; Visser et al., Mol.Gen. Genet., 225, (1991), 289-296), so that this enzyme is ascribed acrucial role in the synthesis of amylose starch. Nucleic acid moleculeswhich code for GBSSI proteins have been described for numerous plants,for example corn (Genbank Acc. No. AF079260, AF079261), wheat (GenbankAcc. No. AB019622, AB019623, AB019624), rice (Genbank Acc. No. AF092443,AF092444, AF031162), potatoes (Genbank Acc. No. X58453), barley (GenbankAcc. No. X07931, X07932). With the aid of these known nucleic acidmolecules, it is possible for the person skilled in the art to isolatecorresponding sequences from other organisms, in particular plantorganisms, by standard processes, for example by heterologous screening.

In the context of the present invention, a branching enzyme or BEprotein (α-1,4-glucan: α-1,4-glucan 6-gycosyltransferase, E.C. 2.4.1.18)is understood as meaning a protein which catalyzes a transglycosylationreaction in which α-1,4-linkages of an α-1,4-glucan donor are hydrolyzedand the α-1,4-glucan chains liberated are transferred to an α-1,4-glucanacceptor chain and converted here into α-1,4 linkages, preferably a BEIprotein.

The term “BEI protein” designates a branching enzyme (BE) of isoform I.The designation of the isoforms follows the nomenclature proposed bySmith-White and Preiss, (Smith-White & Preiss, Plant Mol. Biol. Rep. 12,(1994), 67-71, Larsson et al., Plant Mol. Biol. 37, (1998), 505-511). Inconnection with the present invention, all enzymes which arestructurally more similar to the BEI protein from corn (Baba et al.,Biochem. Biophys. Res. Commun. 181 (1), (1991), 87-94; Kim et al. Gene216, (1998), 233-243), i.e. at the level of the amino acid sequence,than the BEII isoform of the protein from corn (Genbank Acc. NoAF072725, U65948) should be designated as isoform 1. In potato plants,the BEI gene is expressed mainly in the tubers and barely in the leaves(Larsson et al., Plant Mol. Biol. 371 (1998), 505-511).

Nucleic acid molecules which code for a BEI protein have been describedfor numerous plants, for example for corn (Genbank Acc. No. D 11081, AF072724), rice (Genbank Acc. No. D11082), potatoes (various forms of theBEI gene (protein) from potato have been described, for example, inKhoshnoodi et al., Eur. J. Biochem. 242 (1), 148-155 (1996); GenbankAcc. No. Y 08786, Kossmann et al., Mol. Gen. Genet. 230, (1991), 39-44),peas (Genbank Acc. No. X80010). With the aid of these known nucleic acidmolecules, it is possible for the person skilled in the art to isolatecorresponding sequences from other organisms, in particular plantorganisms, by standard processes, for example by heterologous screening.

The present invention further relates to transgenic plant cells whichare genetically modified, the genetic modification consisting in theintroduction of one or more foreign nucleic acid molecules, whosepresence and/or expression leads to a decrease in the activity of GBSSIand BE proteins, in comparison to corresponding non genetically modifiedplant cells of wild-type. plants.

The production of plant cells of this type according to the inventionhaving a decreased activity of a GBSSI and of a BE protein can beacheived by means of various procedures known to the person skilled inthe art, e.g. by means of those which lead to inhibition of theexpression of endogenous genes which encode a GBSSI protein or a BEprotein. These include, for example, the expression of a correspondingantisense RNA, the provision of molecules or vectors which mediate acosuppression effect, the expression of a correspondingly constructedribozyme which specifically cleaves transcripts which encode a GBSSIprotein or a BE protein, or so-called “in-vivo mutagenesis”. All theseprocedures are based on the introduction of one or more foreign nucleicacid molecules into the genome of plant cells.

The term “foreign nucleic acid molecule” is understood as meaning anucleic acid molecule of the type which either does not occur naturallyin corresponding plant cells, or which, in the actual spatialarrangement, does not occur naturally in the plant cells or which islocated at a site in the genome of the plant cell in which it does notoccur naturally. The foreign nucleic acid molecule is preferably arecombinant nucleic acid molecule which consists of various elementswhose combination or specific spatial arrangement does not occurnaturally in plant cells.

The “foreign nucleic acid molecule” can be, for example, a so-called“double construct”, by which is understood a single vector for planttransformation which contains both the genetic information for theinhibition of the expression of one or more endogenous GBSSI genes andfor the inhibition of the expression of one or more BE genes or whosepresence and/or expression leads to a decrease in the activity of one ormore GBSSI proteins and one or more BE proteins.

In a further embodiment of the invention, not only a single specificvector, but a number of different foreign nucleic acid molecules, isintroduced into the genome of the plant cell, one of these foreignnucleic acid molecules being a DNA molecule which, for example, is acosuppression construct which brings about a decrease in the expressionof endogenous GBSSI genes, and a further foreign nucleic acid moleculebeing a DNA molecule which, for example, encodes an antisense RNA whichbrings about a decrease in the expression of endogenous BE genes. Inprinciple, in the construction of the foreign nucleic acid molecules,however, the use of any combination of antisense, cosuppression andribozyme constructs or in-vivo mutagenesis which leads, in thegenetically modified plant cell, to a simultaneous decrease in the geneexpression of endogenous GBSSI and BE genes or which leads to asimultaneous decrease in the activity of GBSSI and BE proteins issuitable.

The foreign nucleic acid molecules can here be introduced into thegenome of the plant cell simultaneously (“cotransformation”) orsuccessively, i.e. chronologically one after the other(“supertransformation”). A number of foreign nucleic acid molecules canbe present in combined form, for example, in a “double construct”.

In one embodiment of the invention, at least one antisense RNA isexpressed in plant cells for the reduction of the activity of one ormore GBSSI proteins and/or one or more BE proteins.

For this, for example, a DNA molecule can be used which comprises theentire sequence coding for a GBSSI protein and/or a BE protein includingflanking sequences, if present, and DNA molecules which only compriseparts of the coding sequence, these parts being long enough to bringabout an antisense effect in the cells. In general, suitable sequencesare those up to a minimum length of 15 bp, preferably of a length of100-500 bp, for an efficient antisense-inhibition, in particularsequences having a length of over 500 bp. As a rule, DNA molecules areused for this which are shorter than 5000 bp, preferably sequences whichare shorter than 2500 bp.

Also possible is the use of DNA sequences which have a high degree ofhomology to the sequences occurring endogenously in the plant cell,which encode a GBSSI protein or a BE protein. The minimal homologyshould be greater than about 65%. The use of sequences having homologiesbetween 95 and 100% is preferred.

In a further embodiment, the decrease in the GBSSI and/or the BEactivity in the plant cells is achieved by a cosuppression effect. Theprocedure is known to the person skilled in the art and is described,for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebelet al., (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell etal. (Curr. Top. Microbiol. Immunol. 197 (1995), 4346), Palaqui andVaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al.,(Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al; (Mol. Gen.Genet. 243 (1994), 613-621). As in the case of the antisense technology,both DNA molecules which code for the entire coding region of the GBSSIand/or of the BE protein, and DNA molecules which comprise only parts ofthe coding sequence, can be used.

Also suitable is the use of DNA sequences which have a high degree ofhomology to the sequences occurring endogenously in the plant cell,which encode GBSSI and/or BE proteins. The minimal homology should begreater than about 65%. The use of sequences with homologies between 95and 100% is preferred. For example, for the inhibition of the BEI genefrom potatoes, a DNA sequence coding for a BEI protein is preferablyused, in particular from potatoes, Kossmann et al. (Mol. Gen. Genet.230, (1991), 3944).

The expression of ribozymes for decreasing the activity of certainenzymes in cells is known to the person skilled in the art and isdescribed, for example, in EP-BI 0321201. The expression of ribozymes inplant cells has been described, for example, in Feyter et al. (Mol. Gen.Genet. 250, (1996), 329-338).

The decrease in the GBSSI and/or the BE activity in the plant cellsaccording to the invention can furthermore also be achieved by so-called“in vivo mutagenesis”, in which a hybrid RNA/DNA oligonucleotide(“chimeroplast”) is inserted into cells by means of cell transformation(Kipp, P. B. et al., Poster Session at the 5^(th) International Congressof Plant Molecular Biology, Sep. 21 st-27th, 1997, Singapore; R. A.Dixon and C. J. Amtzen, Meeting report in “Metabolic Engineering inTransgenic Plants”, Keystone Symposia, Copper Mountain, Colo., USA,TIBTECH 15, (1997), 441447; International Patent Application WO9515972-A1; Kren et al., Hepatology 25, (1997), 1462-1468; Cole-Strausset al., Science 273, (1996), 1386-1389).

A part of the DNA component of the RNA/DNA oligonucleotide is homologousto a nucleic acid sequence of an endogenous GBSSI gene and/or BE gene,but in comparison to the nucleic acid sequence of the endogenous GBSSIgene and/or BE gene has a mutation or contains a heterologous regionwhich is enclosed by the homologous regions.

By means of base pairing of the homologous regions of the RNA/DNAoligonucleotide and of the endogenous nucleic acid molecule, followed byhomologous recombination, the mutation or heterologous region containedin the DNA component of the RNA/DNA oligonucleotide can be transferredto the genome of a plant cell. This leads to a decrease in the activityof a GBSSI protein and/or of a BE protein. It is further known to theperson skilled in the art that it can achieve the activity of a GBSSIprotein and/or of a BE protein by the expression of nonfunctionalderivatives, in particular trans-dominant mutants of such proteinsand/or by the expression of antagonists/inhibitors of such proteins.Antagonists/inhibitors of such proteins include, for example,antibodies, antibody fragments or molecules having similar bindingproperties. For example, a cytoplasmic scFv antibody was employed inorder to modulate the activity of the phytochrome A protein ingenetically modified tobacco plants (Owen, Bio/Technology 10 (1992),790-4; Review: Franken, E, Teuschel, U. and Hain, R., Current Opinion inBiotechnology 8, (1997), 411416; Whitelam, Trends Plant Sci. 1 (1996),268-272).

The present invention therefore relates to transgenic plant cells havingan activity of endogenous GBSSI and BE proteins which is decreased incomparison to unmodified plant cells and which contain one or moreforeign nucleic acid molecules, selected from the group consisting of

-   a) a DNA molecule which leads to the synthesis of at least one    antisense RNA which brings about a decrease in the expression of    endogenous genes which encode a GBSSI and/or a BE protein;-   b) a DNA molecule which leads, via a cosuppression effect, to a    decrease in the expression of endogenous genes which encode a GBSSI    and/or a BE protein;-   c) a DNA molecule which leads to the synthesis of at least one    ribozyme which specifically cleaves transcripts of genes which    encode a GBSSI and/or a BE protein; and-   d) a nucleic acid molecule which, on account of in-vivo mutagenesis,    leads to a mutation or insertion of a heterologous nucleic acid    sequence into at least one gene encoding endogenous GBSSI and/or BE    protein, the mutation or insertion bringing about a decrease in the    expression of the GBSSI gene and/or of the BE gene, or the synthesis    of an inactive GBSSI and/or inactive BE protein.

The expression “decrease in the activity” in the context of the presentinvention means a decrease in the expression of endogenous genes whichencode a GBSSI and a BE protein, a decrease in the amount of GBSSI andBE protein in the cells and/or a decrease in the enzymatic activity ofthe GBSSI and of the BE protein in the cells.

The decrease in the expression can be determined, for example, bymeasurement of the amount of transcripts encoding GBSSI and BE protein,e.g. by Northern blot analysis. A decrease in this case preferably meansa decrease in the amount of transcripts in comparison to correspondingnon genetically modified cells by at least 50%, preferably by at least70%, particularly preferably by at least 85% and very particularlypreferably by at least 95%.

The decrease in the amount of GBSSI and BE protein can be determined,for example, by Western blot analysis. A decrease in this casepreferably means a decrease in the amount of GBSSI and BE protein incomparison to corresponding non genetically modified cells by at least50%, preferably by at least 70%, particularly preferably by at least 85%and very particularly preferably by at least 95%.

The decrease in the enzymatic activity of the GBSSI protein can bedetermined, for example, by the method described by Kuipers et al.,Plant Mol. Biol., 26 (1994), 1759-1773. The decrease in the enzymaticactivity of the BE protein can be determined by the method described bySafford et al., Carbohydrate Polymers 35, (1998), 155-168. A decrease inthe enzymatic activity in comparison to corresponding non geneticallymodified cells in this case preferably means a decrease by at least 50%,preferably by at least 70%, particularly preferably by at least 85% andvery particularly preferably by at least 95%.

The transgenic plant cells according to the invention synthesize amodified starch whose physicochemical properties, for example, inparticular the amylose/amylopectin ratio, the degree of branching, theaverage chain length, the phosphate content, the viscosity behavior, thestarch granule size and/or the starch granule form can be modified incomparison to starch synthesized in wild-type plants such that this isbetter suited to specific intended uses.

It has surprisingly been found that in the case of plant cells in whichthe activity of the GBSSI and of the BEI protein is decreased, thecomposition of the starch is modified in such a manner that it comprisesnot only an amylopectin content of at least 90%, but additionally alsoan increased phosphate content in comparison to plant cells ofcorresponding plants of the waxy phenotype. This increased phosphatecontent acts on the functional properties of the starch such that thisis better suited to specific intended uses.

The present invention also relates to transgenic plant cells whichcontain a modified starch having an amylopectin content of at least 90%,preferably of at least 93%, particularly preferably of at least 95% andespecially preferably of at least 97% and, in comparison to starch fromplant cells of corresponding plants of the waxy phenotype, have anincreased phosphate content.

The amylopectin content can in this case be determined by the method ofHovenkamp-Hermelink et al. (Potato Research 31, (1988), 241-246)described in the examples for potato starch. This method is alsoapplicable to isolated starches of other plant species. Procedures forthe isolation of starches are known to the person skilled in the art.

The expression “increased phosphate content” in connection with thepresent invention means that the total content of covalently bondedphosphate and/or the content of phosphate in the C-6 position of thestarch synthesized in the plant cells according to the invention isincreased by at least 30%, preferably by at least 50%, particularlypreferably by at least 75%, in particular by at least 100% in comparisonto plant cells of corresponding plants of the waxy phenotype.

The total phosphate content or the content of phosphate in the C-6position can be determined by the method described below.

The expression “corresponding plants of the waxy phenotype” is intendedin connection with the present invention to mean comparable plants,preferably plants of the same original variety, i.e. the variety fromwhich the transgenic plants according to the invention have beenproduced by introduction of the genetic modification described above.Plants of the waxy phenotype furthermore contain plant cells whichsynthesize a starch having an amylopectin content of at least 90%,preferably of at least 93%, particularly preferably of at least 95% andespecially preferably of at least 97%. In addition, plants of the waxyphenotype have a decreased enzymatic activity of the GBSSI protein incomparison to plant cells of wild-type plants.

In a further embodiment, the present invention also relates totransgenic plant cells which contain a modified starch having anamylopectin content of at least 90%, preferably of at least 93%,particularly preferably of at least 95% and especially preferably of atleast 97%, have an increased phosphate content in comparison to starchfrom plant cells of corresponding plants of the waxy phenotype and/orhave a decreased gelatinization temperature in comparison to starchesfrom plant cells or corresponding plants of the waxy phenotype.

The gelatinization temperature is intended in the context of the presentinvention to mean the temperature T which can be determined from aviscosity profile (see FIG. 1) which can be obtained by means of a RapidVisco Analyzer (RVA) (Newport Scientific Pty Ltd, Investment SupportGroup, Warriewood, NSW 2102, Australia). The viscosity profile is inthis case plotted according to the protocol described below. Thegelatinization temperature designates that temperature at which theviscosity begins to increase significantly on account of the swelling ofthe starch granules. The gelatinization temperature is determined bymeans of the gradient of the viscosity curve as a function of time. Ifthe gradient of the curve is greater than 1.2 (hits value is specifiedby the user on the RVA apparatus), the computer program identifies thetemperature measured at this time as the gelatinization temperature.

The term “decreased gelatinization temperature” in this case means thatthe gelatinization temperature is decreased by at least 0.5° C.,preferably by at least 1.5° C., particularly preferably by at least 3°C. and especially preferably by at least 5° C. in comparison to starchesfrom plant calls of corresponding plants of the waxy phenotype.

The observation that the starches of the plant cells according to theinvention have a decreased gelatinization temperature in comparison tostarches from plant cells of corresponding plants of the waxy phenotypeis therefore particularly surprising, because Safford et al.(Carbohydrate Polymers 35, (1998), 155-168) were able to show that aphosphate content which was increased in comparison to starches fromwild-type plants, which can be produced by an antisense inhibition of aBEI protein in potato plants, leads to starches having an increasedviscosity onset temperature.

The viscosity onset temperature is a variable related to thegelatinization temperature, which in contrast to the gelatinizationtemperature is based on the method of differential scanning calorimetry(=DSC).

A large number of techniques are available for the introduction of DNAinto a plant host cell. These techniques include the transformation ofplant cells with T DNA using Agrobacterium tumefaciens or Agrobacterumrhizogenes as transforming agents, the fusion of protoplasts, injection,the electroporation of DNA, the incorporation of the DNA by means of thebiolistic approach and other possibilities.

The use of the agrobacteria-mediated transformation of plant cells hasbeen intensively investigated and adequately described in EP-A-120516;Hoekema, in: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., CGt. Rev. Plant Sci.4, 1-46 and An et al. EMBO J. 4, (1985), 277-287. For the transformationof potatoes, see, for example, Rocha-Sosa et al., EMBO J. 8, (1989),29-33).

The transformation of monocotyledonous plants by means of vectors basedon Agrobacterium has also been described (Chan et al., Plant Mol. Biol.22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271-282; Deng etal., Science in China 33, (1990), 28-34; Wilmink et al., Plant CellReports 11, (1992), 7680; May et al., Bio/Technology 13, (1995), 486492;Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie etal., Transgenic Res. 2, (1993), 252-265). An alternative system for thetransformation of monocotyledonous plants is transformation by means ofthe biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994),37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al.,Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl.Genet. 79, (1990), 625-631), protoplast transformation, theelectroporation of partially permeabilized cells, and the incorporationof DNA by means of glass fibers. In particular, the transformation ofcorn is repeatedly described in the literature (cf., for example, WO95106128, EP 0513849, EP 0465875, EP 292435; Fromm et al., Biotechnology8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618;Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor.Appl. Genet. 80, (1990), 721-726).

The successful transformation of other types of cereal has also alreadybeen described, e.g. for barley (Wan and Lemaux, supra; Ritala et al.,supra.; Krens et al., Nature 296, (1982), 72-74) and for wheat (Nehra etal., Plant J. 5, (1994), 285-297).

Generally, any promoter which is active in plant cells is suitable forthe expression of the foreign nucleic acid molecule(s). In this case,the promoter can be chosen such that expression takes placeconstitutively in the plants according to the invention or only in acertain tissue, at a certain point in time of plant development or at apoint in time determined by external influences. With respect to theplant, the promoter can be homologous or heterologous.

Useful promoters are, for example, the promoter of the 35S RNA ofcauliflower mosaic virus and the ubiquitin promoter from corn forconstitutive expression, the patatin gene promoter B33 (Rocha-Sosa etal., EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoesor a promoter which ensures expression only in photosynthetically activetissues, e.g. the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad.Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451), the Ca/b promoter (see, for example, U.S. Pat. No.5,656,496, U.S. Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad.Sci. USA 89, (1992), 3654-3658) and the Rubisco SSU promoter (see, forexample, U.S. Pat. No. 5,034,322, U.S. Pat. No. 4,962,028) or forendosperm-specific expression of the glutelin promoter (Leisy et al.,Plant Mol. Biol. 14, (1990), 41-50; Zheng et al., Plant J. 4, (1993),357-366; Yoshihara et al., FEBS Lett. 383, (1996), 213218), theShrunken-1 promoter (Werr et al., EMBO J. 4, (1985), 1373-1380), the HMGpromoter from wheat, the USP promoter, the phaseolin promoter orpromoters of zein genes from corn (Pedersen et al., Cell 29, (1982),1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).

The expression of the foreign nucleic acid molecule(s) is particularlyof advantage in those organs of the plant which store starch. Suchorgans are, for example, the tuber of the potato plant or the granulesor the endosperm of corn, wheat or rice plants. Promoters are thereforepreferably used which mediate the expression in these organs.

However, promoters can also be used which are only activated at a pointin time determined by external influences (see, for example, WO93107279-A1). Of particular interest in this case can be promoters ofheat-shock proteins which allow simple induction. Seed-specificpromoters, such as, for example, the USP promoter from Vicia faba, whichguarantees seed-specific expression in Vicia faba and other plants(Fiedler et al., Plant Mol. Biol. 22, (1993), 6694679; Bäumlein et al.,Mol. Gen. Genet. 225, (1991), 459-467), can furthermore [lacuna].Fruit-specific promoters can also be employed, such as described, forexample, in WO 91/01373-A1.

A termination sequence can furthermore be present which serves for thecorrect ending of transcription and the addition of a poly-A tail to thetranscript, which is attributed a function in the stabilization of thetranscripts. Elements of this type are described in the literature (cf.,for example, Gielen et al., EMBO J. 8 (1989), 23-29) and are arbitrarilyexchangeable.

The plant cells according to the invention can belong to any desiredplant species, i.e. both to monocotyledonous and to dicotyledonousplants. Preferably, they are plant cells from useful agriculturalplants, i.e. from plants which are cultured by man for food purposes orfor technical, in particular industrial, purposes. The inventionpreferably relates to fiber-forming (e.g. flax, hemp, cotton),oil-storing (e.g. oilseed rape, sunflower, soybeans), sugar-storing(e.g. sugar beet, sugar cane, sorghum) and protein-storing plants (e.g.leguminous plants).

In a further preferred embodiment, the invention relates to feed plants(e.g. feed and pasture grasses (alfalfa, clover etc.)), and vegetableplants (e.g. tomato, lettuce, chicory).

In a particularly preferred embodiment, the invention relates to plantcells from starch-storing plants (e.g. wheat, barley, oats, rye,potatoes, corn rice, peas, cassava); plant cells from potato areparticularly preferred.

The plant cells according to the invention can be used for theregeneration of whole plants.

The plants obtainable by regeneration of the transgenic plant cellsaccording to the invention are likewise the subject of the presentinvention. The present invention further relates to plants which containthe transgenic plant cells described above. The transgenic plants can inprinciple be plants of any desired plant species, i.e. bothmonocotyledonous and dicotyledonous plants. They are preferably usefulplants, i.e. plants which are cultivated by man for food purposes or fortechnical, in particular industrial, purposes. The invention preferablyrelates to plant cells from fiber-forming (e.g. flax, hemp, cotton),oil-storing (e.g. oilseed rape, sunflower, soybeans)), sugar-storing(e.g. sugar beet, sugar cane, sorghum) and protein-storing plants (e.g.leguminous plants).

In a further preferred embodiment, the invention relates to feed plants(e.g. feed and pasture grasses (alfalfa, clover etc.) and vegetableplants (e.g. tomato, lettuce, chicory).

In a particularly preferred embodiment the invention relates tostarch-storing plants (e.g. wheat, barley, oats, rye, potatoes, corn,rice, peas, cassava); potato plants are particularly preferred.

The present invention also relates to a process for the production of atransgenic plant cell or plant which synthesizes a modified starch,where

-   (a) a plant cell is genetically modified by the introduction of one    or more foreign nucleic acid molecules, whose presence and/or    expression lead/leads to a decrease in the activity of a protein    having the activity of a GBSSI protein and to a decrease in the    activity of a protein having the activity of a BE protein; and in    the case of the production of a plant-   (b) a plant is regenerated from the cell produced according to step    a); and, if appropriate, further plants are produced from the plants    produced according to step b).

The present invention also relates to a process for the production of atransgenic plant cell or plant whose starch has an amylopectin contentof at least 90% and an increased phosphate content in comparison tostarch from corresponding plants of the waxy phenotype, where

-   (a) a plant cell is genetically modified by the introduction of one    or more foreign nucleic acid molecules, whose presence or whose    expression leads to a decrease in the activity of a protein having    the activity of a GBSSI protein and to a decrease in the activity of    a protein having the activity of a BEI protein;    and in the case of the production of a plant-   (b) a plant is regenerated from the cell produced according to step    a); and, if appropriate, further plants are produced from the plants    produced according to step b).

The present invention furthermore relates to a process for theproduction of a transgenic plant cell or plant whose starch has anamylopectin content of at least 90% and which has an increased phosphatecontent and/or a decreased gelatinization temperature T in comparison tostarch from corresponding plants of the waxy phenotype, where

-   (a) a plant cell is genetically modified by the introduction of one    or more foreign nucleic acid molecules whose presence or whose    expression leads to a decrease in the activity of a protein having    the activity of a GBSSI protein and to a decrease in the activity of    a protein having the activity of a BEI protein;    and in the case of the production of a plant-   (b) a plant is regenerated from the cell produced according to a);    and, if appropriate, further plants are produced from the plant    produced according to step b).

The expressions “increased phosphate content” and “decreasedgelatinization temperature” are as already defined above in thisconnection.

For the genetic modification introduced according to step a), the sameapplies as has already been explained above in a different connectionwith the plants according to the invention.

The regeneration of plants according to step b) can be carried out bymethods known to the person skilled in the art.

The production of further plants according to step b) of the processaccording to the invention can be carried out, for example, byvegetative reproduction (for example by means of cuttings, tubers or bymeans of callus culture and regeneration of whole plants) or by sexualreproduction. Sexual reproduction in this case preferably takes place ina controlled manner i.e. selected plants having certain properties arecrossed with one another and reproduced. Of course, it is known to theperson skilled in the art that, for the production of the plant cellsand plants according to the invention, he/she can also use transgenicplants in which the activity of one of the abovementioned proteins hasalready been decreased and which, in accordance with the processaccording to the invention, only have to be genetically modifiedinasmuch as the activity of the second protein is also decreased.

It is furthermore known to the person skilled in the art that thesuper-transformation described above is not necessarily carried out inprimary transformants, but preferably in previously selected stabletransgenic plants which advantageously have already been tested by meansof appropriate experiments for, for example, fertility, stableexpression of the foreign gene, hemi- and homozygotism etc.

The present invention also relates to the plants obtainable by theprocess according to the invention.

The present invention also relates to reproductive material of plantsaccording to the invention and to the transgenic plants produced inaccordance with the process according to the invention. The termreproductive material in this case includes those constituents of theplants which are suitable for the production of descendants by avegetative or generative route. For vegetative reproduction, thosesuitable are, for example, cuttings, callus cultures, rhizomes ortubers. Other reproductive material includes, for example, fruits,seeds, seedlings, protoplasts, cell cultures, etc. Preferably, thereproductive material comprises tubers and seeds.

The present invention furthermore relates to the use of one or moreforeign nucleic acid molecules which encode a protein having theenzymatic activity of a GBSSI protein and a protein having the enzymaticactivity of a BE protein or the use of fragments of said nucleic acidmolecules for the production of plant cells or plants which synthesize amodified starch.

In a further preferred embodiment, the present invention relates to theuse of one or more foreign nucleic acid molecules which encode(s) aprotein having the enzymatic activity of a GBSSI protein and a proteinhaving the enzymatic activity of a BEI protein or the use of fragmentsof said nucleic acid molecule(s) for the production of plants whichsynthesize a modified starch which, in comparison to starch fromcorresponding plants of the waxy-phenotype, has an increased phosphatecontent and/or a decreased gelatinization temperature.

The term “fragment(s)” in this connection is intended to mean a part ofthe foreign nucleic acid molecule(s) which can code, for example, for afunctionally active part of the proteins described. The fragment canfurthermore also code for an antisense or a cosuppression mRNA or for aribozyme. When using the fragments, care must be taken that only thosefragments are used which lead to a decrease in the enzymatic activity ofa GBSSI and/or of a BE or of a BEI protein.

In a further embodiment, the present invention relates to the use of oneor more foreign nucleic acid molecules for the production of plantswhich synthesize a starch having an amylopectin content of at least 90%,which, in comparison to starch from corresponding plants of the waxyphenotype, has an increased phosphate content and/or a decreasedgelatinization temperature, the foreign nucleic acid molecule being amolecule or the foreign nucleic acid molecules being a number ofmolecules selected from the group consisting of:

-   a) DNA molecules which encode at least one antisense RNA which    brings about a decrease in the expression of endogenous genes    encoding GBSSI and/or BEI proteins;-   b) DNA molecules which via a cosuppression effect lead to a decrease    in the expression of endogenous genes encoding GBSSI and/or BEI    proteins;-   c) DNA molecules which encode at least one ribozyme which    specifically cleaves transcripts of endogenous genes encoding GBSSI    and/or BEI proteins; and-   d) nucleic acid molecules introduced by means of in-vivo    mutagenesis, which lead to a mutation or an insertion of a    heterologous sequence in one or more endogenous GBSSI and/or BEI    proteins, the mutation or insertion bringing about a decrease in the    expression of the GBSSI gene and/or the BEI gene or the synthesis of    an inactive GBSSI protein and/or an inactive BEI protein.

As already explained above, the foreign nucleic acid molecules can beintroduced into the genome of the plant cell simultaneously or elsesuccessively. In this case, the simultaneous introduction of the foreignnucleic acid molecules is time- and cost-saving, i.e. cotransformationin which, in a transformation experiment in accordance with the processaccording to the invention described above, (a) nucleic acid molecule(s)is/are preferably introduced into the plant cell, whose presence and, ifappropriate, expression leads/lead to a decrease in the activity of aprotein having the activity of a GBSSI protein and to a decrease in theactivity of a protein having the activity of a BE protein, preferably ofa BEI protein. The present invention therefore also relates tocompositions which contain at least one of the nucleic acid moleculesdescribed above. Preferably, heir introduction into plant cells leads toa decrease in the activity of a GBSSI protein and to a decrease in theactivity of a protein having the activity of a BE protein, preferably ofa BEI protein. In this case, in the composition according to theinvention the nucleic acid molecules whose respective presence in theplant cell leads to a decrease in the activities of GBSSI and of BEproteins, preferably of a BEI protein, can be included either separatelyor together in a recombinant nucleic acid molecule. In the first case,the composition according to the invention can contain, for example, twoor more recombinant vectors whose joint presence in the plant cell leadsto said phenotype. In the second case, which is preferred according tothe invention, a recombinant nucleic acid molecule contains the geneticinformation which leads to a decrease in the activity of a GBSSI and ofa BE protein, preferably of a BEI protein. For example, in such arecombinant nucleic acid molecule the nucleic acid molecules describedabove, whose presence in a plant cell leads to a decrease in theactivity of a GBSSI or of a BE or BEI protein, can be present as achimeric gene or as separate genes. Numerous examples of such double ormultiple constructs are described in the technical literature. Theaforementioned recombinant nucleic acid molecules can be present in anydesired host cell which thereby is likewise a subject of the presentinvention. A further advantage in the use of such double or multipleconstructs is that plant cells and plants according to the invention canbe identified more easily, for example by means of an appropriate choiceof PCR primers or Southern blots. The plant cells and plants accordingto the invention are therefore preferably characterized by the presenceof such double or multiple constructs.

On account of the expression of a foreign nucleic acid molecule or of anumber of foreign nucleic acid molecules whose presence or whoseexpression leads to a decrease in the activity of a GBSSI protein and toa decrease in the activity of a BE protein, preferably of the BEIprotein, in comparison to corresponding non genetically modified plantcells of wild-type plants, the transgenic plant cells and plantsaccording to the invention synthesize a starch whose physicochemicalproperties, in particular the amylose/amylopectin ratio and/or thephosphate content and/or the gelatinization behavior, are modified incomparison to starch synthesized in wild-type plants.

The present invention therefore relates to starch which is obtainablefrom the transgenic plant cells, plants and reproductive materialaccording to the invention.

Furthermore, the present invention also relates to starches which havean amylopectin content of at least 90% and a phosphate content which isincreased in comparison to starch from corresponding plants of the waxyphenotype by at least 30%, preferably by at least 50%, particularlypreferably by at least 75%, in particular by at least 100%, incomparison to plant cells of corresponding plants of the waxy phenotype.

On account of the increased phosphate content, the starches according tothe invention have the advantage over conventional waxy starches thatthey are better suited to certain intended uses on account of theirmodified physicochemical properties. On account of the increasedphosphate content, for this the starches according to the inventionsubsequently do not have to be or have to be less strongly chemicallyphosphorylated in comparison to conventional waxy starches.

The starches according to the invention preferably differ fromchemically phosphorylated monophosphate waxy starches in theirstructural/functional properties.

Phosphorylated waxy starches are particularly suitable for use for allthickenings without a skin or gel formation in the desserts, delicaciesand ready-to-serve meals area, especially also for deep-freeze products.In technical areas, monostarch phosphates are in some cases used forpaper manufacture, additionally as sizing, flocculating and flotationagents and as a detergent additive.

In a further embodiment, the present invention also relates to starcheswhich have an amylopectin content of at least 90%, a phosphate contentwhich is increased in comparison to starch from corresponding plants ofthe waxy phenotype by at least 30% and/or a decreased gelatinizationtemperature T.

The term “decreased gelatinization temperature” in this connection isintended as having the meaning already defined above.

In connection with certain applications and technical processes, adecreased gelatinization temperature allows the saving of heat energyand/or simplified process implementation.

In a particularly preferred embodiment, the present invention relates tostarches from potato plants which have an amylopectin content of atleast 90%, a phosphate content which is increased in comparison tostarch from corresponding plants of the waxy phenotype by at least 30%and/or a decreased gelatinization temperature T.

In comparison to native starch from corn, rice or wheat plants, nativepotato starches have an increased phosphate content, which predestinesthem for certain applications. Surprisingly, it is possible with the aidof the approach according to the invention to increase the phosphatecontent of waxy potato starches further so that the potato starchesaccording to the invention, in comparison to potato starches fromcorresponding wild-type plants and/or in comparison to correspondingpotato plants of the waxy phenotype, have a phosphate content which isincreased by at least 30%, preferably by at least 50%, particularlypreferably by at least 75% and in particular by at least 100%. Potatostarches furthermore have the advantage over cereal starches (e.g.wheat, oats, corn, rice) that they have a low content of lipids andproteins.

In a further preferred embodiment of the invention, the potato starchesare characterized by an amylopectin content of at least 93%,particularly preferably of at least 95% and especially preferably of atleast 97% and/or by a phosphate content which is increased in comparisonto starch from plant cells of corresponding plants of the waxy phenotypeby at least 30%, preferably by at least 50%, particularly preferably byat least 75%, in particular by at least 100%, in comparison to plantcells of corresponding plants of the waxy phenotype and/or by agelatinization temperature which is decreased in comparison to starchfrom plant cells of corresponding plants of the waxy phenotype by atleast 0.5° C., preferably by at least 1.5° C., particularly preferablyby at least 3° C. and especially preferably by at least 5° C.

In this connection, the terms “phosphate content” and “gelatinizationtemperature” are defined in the same manner as already described above.

The present invention furthermore relates to a process for theproduction of a modified starch comprising the step of extraction of thestarch from a plant (cell) according to the invention described aboveand/or from starch-storing parts of such a plant. Preferably, such aprocess also includes the step of the harvesting of the cultivatedplants and/or starch-storing parts of these plants before the extractionof the starch and particularly preferably furthermore the step of thecultivation of plants according to the invention before harvesting.Processes for the extraction of the starch from plants or fromstarch-storing parts of plants are known to the person skilled in theart. Processes for the extraction of the starch from various otherstarch-storing plants are furthermore described, e.g. in “Starch:Chemistry and Technology (ed.: Whistler, BeMiller and Paschall (1994),2nd edition, Academic Press Inc. London Ltd; ISBN 0-12-746270-8; see,for example, chapter XII, pages 412-468: corn and sorghum starches:production; by Watson; chapter XIII, pages 469-479: tapioca, arrowrootand sago starches: production; by Corbishley and Miller; chapter XIV,pages 479-490: potato starch: production and uses; by Mitch; chapter XV,pages 491 to 506: wheat starch: production, modification and uses; byKnight and Oson; and chapter XVI, pages 507 to 528: rice starch:production and uses; by Rohmer and Klem; corn starch: Eckhoff et al.,Cereal Chem. 73 (1996) 54-57, as a rule the extraction of corn starch onan industrial scale is achieved by so-called wet-milling). Applianceswhich are usually used in processes for the extraction of starch fromplant material are separators, decanters, hydrocyclones, spray dryersand fluidized bed dryers.

The present invention furthermore relates to starch which is obtainableby the process according to the invention described above.

The starches according to invention can be subsequently modified by theprocesses known to the person skilled in the art and are suitable inunmodified or modified form for various uses in the foodstuffs or nonfoodstuffs area.

Basically, the possibilities of application of the starch can besubdivided into two large areas. One area includes the hydrolysisproducts of the starch, mainly glucose and glucan units, which areobtained by enzymatic or chemical processes. They serve as startingsubstances for further chemical modifications and processes, such asfermentation. For reduction of the costs, the simplicity and inexpensivecarrying-out of a hydrolysis process can be of importance here. Atpresent, it proceeds essentially enzymatically using amyloglucosidase. Asaving of costs by lower use of enzymes would be conceivable. Astructural modification of the starch, e.g. surface enlargement of thegranule, or easier digestibility due to a lower degree of branching or asteric structure which restricts the accessibility for the enzymesemployed could bring this about.

The other area in which starch is used as so-called native starchbecause of its polymeric structure is divided into two further fields ofuse:

1. Foodstuffs Industry

-   -   Starch is a classical additive for many foodstuffs, in which it        essentially takes on the function of the binding of aqueous        additives or causes an increase in the viscosity or else        increased gel formation. Important characteristic features are        the flow and sorption behavior, the swelling and gelatinization        temperature, the viscosity and thickening power, the solubility        of the starch, the transparency and gel structure, the heat,        shear and acid stability, the proneness to retrogradation, the        capacity for film formation, the freezing/thawing stability, the        digestibility and the capacity for complex formation with, for        example, inorganic or organic ions.

2. Non Foodstuffs Industry

-   -   In this wide area, starch can be employed as an auxiliary for        different production processes or as an additive in technical        products. When using starch as an auxiliary, the paper and        cardboard industry is to be mentioned here in particular. Starch        primarily serves here for the retardation (retention of solids),        the binding of filler and fine material particles, as a        stiffener and for dehydration. Moreover, the favorable        properties of the starch are utilized in relation to stiffness,        hardness, sound, grip, gloss, smoothness, bonding strength and        the surfaces.

2.1 Paper and Cardboard Industry

-   -   Within the paper manufacturing process, four application areas,        namely surface, coating, mass and spraying, are to be        differentiated. The demands on the starch in relation to the        surface treatment are essentially a high degree of whiteness, a        suitable viscosity, a high viscosity stability, good film        formation and low dust formation. When used in coating, the        solids content, a suitable viscosity, a high binding power and a        high pigment affinity play an important role. As an additive to        the mass, a rapid, uniform, loss-free distribution, high        mechanical stability and complete retention in the paper web are        of importance. When the starch is used in the spraying area, a        suitable solids content, high viscosity and a high binding power        are likewise of importance.

2.2 Adhesives Industry

-   -   A wide area of use of starches is in the adhesives industry,        where the possibilities of use can be divided into four        subareas: the use as pure starch size, the use in starch sizes        prepared using special chemicals, the use of starch as an        additive to synthetic resins and polymer dispersions, and the        use of starches as extenders for synthetic adhesives. 90% of the        adhesives based on starch are employed in the areas corrugated        cardboard production, production of paper sacks, sachets and        bags, production of composite materials for paper and aluminum,        production of cardboard boxes and gum for envelopes, stamps etc.

2.3 Textile and Textile Care Compositions Industry

A wide area of use for starches as auxiliaries and additives is theproduction of textiles and textile care compositions area. Within thetextile industry, the following four areas of use can be differentiated:the use of starch as a sizing agent, i.e. as an auxiliary for smoothingand strengthening the clinging behavior for protection against thetensile forces acting during weaving, and for increasing the abrasionresistance during weaving, starch as an agent for textile finishing,especially after quality-impairing pretreatments, such as bleaching,dyeing etc., starch as a thickener in the production of dye pastes forthe prevention of dye diffusion, and starch as an additive to warpingagents for sewing threads.

2.4 Building Materials Industry

-   -   The fourth area of use is the use of starches as additives in        building materials. One example is the production of        plasterboard sheets, in which the starch mixed in the plaster        slurry gelatinizes with the water, diffuses to the surface of        the plaster sheet and there binds the board to the sheet.        Further areas of use are admixture to rendering and mineral        fibers. In the case of ready-mixed concrete, starch products are        used to delay setting.

2.5 Soil Stabilization

-   -   A further market for starch offers itself in the production of        compositions for soil stabilization, which are employed for the        temporary protection of the soil particles against water when        the soil is moved artificially. According to present knowledge,        combination products of starch and polymer emulsions are to be        equated to the products previously employed in their erosion-        and encrustation-decreasing action, but have markedly lower        prices than these.

2.6 Use in Plant Protection Agents and Fertilizers

-   -   One area of use is the use of starch in plant protection agents        for modifying the specific properties of the preparations. Thus        starch can be employed for improving the welting of plant        protection agents and fertilizers, for the metered release of        the active compounds, for the conversion of liquid, volatile        and/or foul-smelling active compounds into microcrystalline,        stable, shapeable substances, for mixing incompatible compounds        and for extending the duration of action by decreasing        decomposition.

2.7 Pharmaceuticals, Medicine and the Cosmetics Industry

-   -   A further area of use is the area of pharmaceuticals, medicine        and the cosmetics industry. In the pharmaceutical industry,        starch can be employed as a binder for tablets or for diluting        the binder in capsules. Furthermore, starch can serve as a        tablet disintegrant, as it absorbs liquid after swallowing and        after a short time swells to such an extent that the active        compound is released. Medicinal lubricating powders and wound        powders are based on starch for reasons of quality. In the        cosmetics area, starches are employed, for example, as carriers        of powder additives, such as fragrances and salicylic acid. A        relatively wide area of application for starch is in toothpaste.

2.8 Addition of Starch to Charcoal and Briquettes

-   -   One area of use of starch is as an additive to charcoal and        briquettes. Charcoal can be agglomerated or made into briquettes        of high grade quantitatively using the addition of starch, by        means of which premature disintegration of the briquette is        prevented. In the case of barbecue charcoal, the addition of        starch is between 4 and 6%; in the case of calorized charcoal        between 0.1 and 0.5%. In addition, starches are gaining        importance as binders, as the emission of harmful substances can        be markedly decreased by means of their addition to charcoal and        briquettes.

2.9 Ore and Coal Slurry Preparation

Starch can furthermore be employed in ore and coal slurry preparation asa flocculant.

2.10 Foundry Auxiliary

-   -   A further area of use is as an additive to foundry auxiliaries.        In various casting processes, cores are needed which are        prepared from sands treated with binders. The binder used today        is mainly bentonite, which is treated with modified starches,        usually swellable starches.    -   The purpose of the addition of starch is increasing the        flowability and improving the adhesiveness. Moreover, the        swellable starches can be subject to further production        engineering demands, such as being dispersible in cold water,        rehydratable, readily miscible in sand and having high water        binding capacity.

2.11 Use in the Rubber Industry

-   -   In the rubber industry, starch can be employed for improving the        technical and optical quality. The reasons in this case are the        improvement of the surface luster, the improvement of the grip        and of the appearance (for this, starch is scattered onto the        tacky gummed surfaces of rubber materials before cold        vulcanization), and also the improvement of the printability of        the rubber.

2.12 Production of Leather Substitutes

-   -   A further marketing possibility for the modified starches is the        production of leather substitutes.

2.13 Starch in Synthetic Polymers

-   -   In the plastics sector, the following areas of use stand out:        the inclusion of starch secondary products in the finishing        process (starch is only a filler, there is no direct bond        between the synthetic polymer and starch) or alternatively the        inclusion of starch secondary products in the production of        polymers (the starch and polymer form a permanent bond).

Compared with the other substances such as talc, the use of starch as apure filler is not competitive. Things are different if the specificproperties of starch come to bear and as a result the character profileof the final products is distinctly modified. An example of this is theuse of starch products in the finishing of thermoplastics, such aspolyethylene. The starch and the synthetic polymer here are combined bycoexpression in the ratio of 1:1 to give a ‘masterbatch’, from whichvarious products can be produced with granulated polyethylene usingconventional process techniques. By the indusion of starch inpolyethylene films, an increased substance permeability in the case ofhollow bodies, an improved water vapor permeability, an improvedantistatic behavior, an improved antiblock behavior and an improvedprintability with aqueous dyes can be achieved.

Another possibility is the use of starch in polyurethane foams. Withadaptation of the starch derivatives and by process engineeringoptimization, it is possible to control the reaction between syntheticpolymers and the hydroxyl groups of these starches specifically. Theresult is polyurethane films which, owing to the use of starch, obtainthe following characteristic profiles: a decrease in the heat expansioncoefficients, decrease in the shrinkage behavior, improvement of thepressure/tension behavior, increase in the water vapor permeabilitywithout modification of water absorption, decrease in the flammabilityand tear density, no dropping of combustible parts, freedom from halogenand decreased aging. Disadvantages which are currently still present aredecreased resistance to pressure and a decreased impact strength.

Product development, meanwhile, is no longer restricted only to films.Solid plastic products, such as pots, plates and dishes, can also beproduced with a starch content of over 50%. In addition, starch/polymermixtures are to be favorably assessed, as they have a very much higherbiodegradability.

Starch graft polymers have furthermore gained extraordinary importanceon account of their extreme water-binding ability. These are productshaving a backbone of starch and a side lattice of a synthetic monomergrafted on according to the principle of the free-radical chainmechanism. The starch graft polymers available today are distinguishedby a better binding and retention capacity of up to 1000 g of water perg of starch combined with high viscosity. The application areas forthese superabsorbers have greatly expanded in recent years and lie inthe hygiene area with products such as diapers and pads, and in theagricultural sector with seed coatings, for example.

What is crucial for the use of the novel, genetically modified starchesis, on the one hand, the structure, water content, protein content,lipid content, fiber content, ash/phosphate content, amylose/amylopectinratio, molar mass distribution, degree of branching, particle size andshape and crystallinity, and on the other hand also the properties whichlead to the following features: flow and sorption behavior,gelatinization temperature, viscosity, thickening power, solubility, gelstructure and transparency, heat, shear and acid stability, proneness toretrogradation, gel formation, freeze/thaw stability, complex formation,iodine binding, film formation, adhesive power, enzyme stability,digestibility and reactivity.

The production of modified starches by means of genetic engineeringinterventions in a transgenic plant can on the one hand modify theproperties of the starch obtained from the plant to the effect thatfurther modifications by means of chemical or physical processes nolonger appear necessary. On the other hand, the starches modified bygenetic engineering processes can be subjected to further chemicaland/or physical modifications, which leads to further improvements inthe quality for certain of the areas of use described above. Thesechemical and physical modifications are known in principle. Inparticular, they are modifications by:

-   -   heat treatment,    -   acid treatment,    -   production of starch ethers        -   starch alkyl ethers, O-allyl ethers, hydroxylalkyl ethers,            O-carboxylmethyl ethers, N-containing starch ethers,            P-containing starch ethers, S-containing starch ethers    -   production of crosslinked starches    -   production of starch graft polymers    -   oxidation and    -   esterifications which lead to the formation of phosphate,        nitrate, sulfate, xanthate, acetate and citrate starches.        Further organic acids can likewise be employed for        esterification.

The figures show:

FIG. 1: Schematic representation of an RVA profile

The following methods were used in the examples:

1. Starch Analysis Methods a) Determination of the Amylose/AmylopectinRatio

-   -   Starch was isolated from potato plants by standard methods, and        the amylose to amylopectin ratio was determined according to the        method described by Hovenkamp-Hermelink et al. (Potato Research        31, (1988), 241-246).

b) Determination of the Phosphate Content

-   -   The positions C2, C3 and C6 of the glucose units can be        phosphorylated in starch. For the determination of the phosphate        group content at the C6 position, 100 mg of starch were        hydrolyzed at 95° C. for 4 hours in 1 ml of 0.7 M. HCl (Nielsen        et al., Plant Physiol. 105, (1994), 11-117). After        neutralization with 0.7 M KOH, 50 μl of the hydrolyzate were        subjected to an optical enzymatic test for glucose 6-phosphate        determination. The change in the absorption of the test batch        (100 mM imidazole/HCl; 10 mM MgCl₂; 0.4 mM NAD; 2 units of        glucose 6-phosphate dehydrogenase from Leuconostoc        mesenteroides; 30° C.) was monitored at 334 nm.    -   The total phosphate content was determined according to the        method of Ames (Methods in Enzymology VIII, (1966), 115-118).    -   About 50 mg of starch are treated with 30 μl of ethanolic        magnesium nitrate solution and incinerated in a muffle furnace        at 500° C. for three hours. The residue is treated with 300 μl        of 0.5 M hydrochloric acid and incubated at 60° C. for 30 min.    -   An aliquot is then made up to 300 μl with 0.5 M hydrochloric        acid, added to a mixture of 100 μl of 10% strength ascorbic acid        and 600 μl of 0.42% ammonium molybdate in 2 M sulfuric acid and        incubated at 45° C. for 20 min.    -   Photometric determination at 820 nm follows taking into account        a phosphate calibration series as the standard.

c) Determination of the Gel Solidity (Texture Analyzer)

-   -   2 g of starch (TS) are gelatinized in 25 ml of H₂O (cf. RVA) and        then stored at 25° C. in a sealed air-tight container for 24 h.        The samples are fixed under the probe (round stamp) of a texture        analyzer TA-XT2 from Stable Micro Systems and the gel solidity        is determined using the following parameters:

test speed 0.5 mm/s penetration depth 7 mm contact area 113 mm² pressure2 g

d) Viscosity Profile

-   -   2 g of starch (TS) are taken up in 25 ml of H₂O and used for        analysis in a Rapid Visco Analyser (Newport Scientific Pty Ltd.,        Investment Support Group, Warriewood NSW 2102, Australia). The        apparatus is operated according to the instructions of the        manufacturer. For determination of the viscosity of the aqueous        solution of the starch, the starch suspension is first heated        from 50° C. to 95° C. at a rate of 12° C. per minute. The        temperature is then kept at 95° C. for 2.5 min. The solution is        then cooled from 95° C. to 50° C. at a rate of 12° C. per        minute. The viscosity is determined during the entire time.    -   The gelatinization temperature is determined by means of the        gradient of the viscosity curve as a function of time. If the        gradient of the curve is greater than 1.2 (this value is        specified by the user), the computer program identifies the        temperature measured at this point in time as the gelatinization        temperature.

e) Determination of Glucose, Fructose and Sucrose

-   -   The content of glucose, fructose and sucrose is determined        according to the method described by Stitt et al. (Methods in        Enzymology 174 (1989), 518-552).

f) Analysis of the Side Chain Distribution of the Amylopectin

-   -   The side chain distribution, resp. length is determined as        described in Lloyd et al., Biochem. J. 338, (1999), 515-521. The        following elution conditions are chosen:

Time min 0.15 M NaOH % 1 M NaAc in 0.15 M NaOH % 0 100 0 5 100 0 20 8515 35 70 30 45 68 32 60 0 100 70 0 100 72 100 0 80 100 0

g) Particle Size Determination

-   -   The particle size determination was carried out using a        photo-sedimentometer of the “Lumosed” type from Retsch GmbH,        Germany.    -   The particle size distribution was determined in an aqueous        solution and was carried out according to the manufacturer's        instructions and based on the literature of, for example, H.        Pitsch, particle size determination; LABO-1988/3 Technical        Journal for Laboratory Technology, Darmstadt

h) Water-Binding Ability

-   -   For the determination of the water-binding ability, the residue        was weighed after the separation of the soluble portion by        centrifugation of the starch swollen at 70° C. The water-binding        ability (WBA) of the starch was related to the initial starch        weight corrected by the soluble mass.

WBA(gig)=(residue−(initial weight−soluble portion)/(initialweight−soluble portion)

The following vectors were used in the examples:Details of the Vector pBinAR-Hyg

The plasmid pBinAR is a derivative of the binary vector plasmid pBin19(Bevan, 1984), which was constructed in the following manner:

a fragment 529 bp long which comprises the nucleotides 6909-7437 of the35S promoter of the cauliflower mosaic virus was isolated as the EcoRI/Kpn I fragment from the plasmid pDH51 (Pietrzak et al., 1986) andligated between the EcoR I and Kpn I cleavage sites of the polylinker ofpUC18. The plasmid pUC18-358 was formed. From the plasmid pAGV40(Herrera-Estrella et al., 1983), with the aid of the restrictionendonucleases Hind III and Pvu II a fragment 192 bp long was isolatedwhich comprises the polyadenylation signal (3′ end) of the octopinsynthase gene (gene 3) of the T DNA of the Ti plasmid pTiACH₅ (Gielen etal., 1984) (nucleotides 11749-11939). After addition of Sph I linkers tothe Pvu II cleavage site, the fragment was ligated between the Sph I andHind III cleavage sites of pUC18-35S. The plasmid pA7 resulted fromthis. Starting from the plasmid pA7, the EcoRI Hind III fragmentcontaining the 35S RNA promoter, the ocs terminator and the part of thepolylinker situated between the 35S RNA promoter and the ocs element wasligated into the appropriately cleaved pBIB-Hyg plasmid (Becker, 1990).

The following examples illustrate the invention without restricting itin any way.

EXAMPLE 1 Production of Transgenic Potato Plants which have a DecreasedActivity of a GBSSI and of a BEI Protein

For the production of transgenic plants which have a decreased activityof a GBSSI and of a BEI protein, transgenic plants were first producedwhich had a decreased GBSSI activity. For this purpose, the T-DNA of theplasmid pB33aGBSSI-Kan was transferred to potato plants with the aid ofagrobacteria, as described in Roch-Sosa et al. (EMBO J. 8, (1989),23-29).

For the construction of the plasmid pB33aGBSSI-Kan, the DraI/DraIfragment from the promoter region of the patatin dass I gene B33 ofSolanum tuberosum, comprising the nucleotides −1512 to +14 (Rocha-Sosaet al., (1989), see above) was ligated into the SmaI cleavage site ofthe plasmid pUC19 (Genbank Acc. No. M77789). From the resulting plasmid,the promoter fragment was ligated as EcoRI/HindIII fragment into thepolylinker region of the plasmid pBin19 (Bevan et al., Nucl Acids Res11, (1983), 369-385). The 3′EcoRI fragment, nucleotides +1181 to +2511of the GBSSI gene from Solanum tuberosum (Hergersberg, Molecularanalysis of the waxy gene from Solanum tuberosum and expression of waxyantisense RNA in potatoes. Dissertation at University of Cologne (1988))was then ligated into the EcoRI cleavage site of the resulting plasmid.The plasmid pB33aGBSSI-Kan resulted.

After the transformation, various lines of transgenic potato plants wereidentified which had a markedly decreased content of the mRNA of a GBSSIand a decreased activity of the GBSSI protein. Plants of this typefurthermore synthesize a starch having an amylopectin content of atleast 90%.

Two independent lines of these amylopectin-synthesizing plants were thentransformed using the plasmid p35SaBEI-Hyg. For the construction of thisplasmid, a SmaI/HindIII fragment about 3000 bp long, containing apartial cDNA for the BEI enzyme from potato (Kossmann, cloning andfunctional analysis of genes coding for proteins involved in thecarbohydrate exchange of the potato, Dissertation Technical UniversityBerlin, (1992)) was smoothed out and inserted in antisense orientationwith respect to the 35S promoter into the SmaI cleavage site of thevector pBinAR-Hyg (see above).

After the supertransformation, various independent lines were identifiedwhich indeed contained the same T-DNAs, but integrated at differentsites in the genome, which had both a decreased GBSSI activity and asignificantly decreased amount of BEI-mRNA. Plants were selected which,in comparison to corresponding wild-type plants, have an enzyme activityof the GBSSI protein reduced by preferably at least 95% and an enzymeactivity of the BEI protein reduced by at least 90%. The starches ofthese plants were then analyzed.

EXAMPLE 2 Analysis of the Starch from Plants having Decreased GBSSI andBEI Activity

The starch formed by the transgenic potato plants produced according toExample 1 differs, for example, from starch synthesized in wild-typeplants in its phosphate or amylose content and in the viscosity andgelatinization properties determined by means of RVA. The results of thephysico-chemical characterization of the modified starches are shown inTable I (Tab. 1).

TABLE 1 Phosphate Total RVA RVA RVA RVA Gel Geno- in C6 phosphateAmylose Max Min Fin Set RVA T solidity No. type (%) (%) (%) (%) (%) (%)(%) (%) (%) 1 Desiree 100 100 22 100 100 100 100 100 100 (wild-type) 2AsGBSSI 110 119 <4 70 90 84 57 104 21 3 AsBEI 170 20 124 94 90 76 100 914 AsGBSSI- 181 189 <4 69 84 78 51 102 21 asBEI Legend: GBSSI =granule-bound starch synthase I BEI = branching enzyme I as = antisenseRVA = Rapid Visco Analyser Max = maximum viscosity Min = minimumviscosity Fin = viscosity at the end of the measurement Set = setback =difference between Min and Fin T = gelatinization temperature With theexception of the amylose content, the % values are based on the wildtype (=100%).

1-29. (canceled)
 30. A method of producing potato starch comprisingextracting starch from a plant comprising transgenic plant cells whichare genetically modified, said genetic modification leading to adecrease in the activity of one or more GBSSI proteins occurringendogenously in the plant cell and to a decrease in the activity of oneor more BE proteins occurring endogenously in the plant cell, incomparison to corresponding wild-type plants.
 31. The method of claim30, further comprising harvesting before the step of extracting.
 32. Themethod of claim 31, further comprising cultivating before the step ofharvesting.
 33. The method of claim 30, wherein said potato starchcomprises an amylopectin content of at least 90% and a phosphate contentwhich is increased in comparison to starch from corresponding potatoplants of the waxy phenotype by at least 30%, wherein said starch fromcorresponding potato plants of the waxy phenotype is derived from theDesiree variety.
 34. A method of producing potato starch comprisingextracting starch from a plant cell which is genetically modified, saidgenetic modification leading to a decrease in the activity of one ormore GBSSI proteins occurring endogenously in the plant cell and to adecrease in the activity of one or more BE proteins occurringendogenously in the plant cell, in comparison to corresponding wild-typeplants.
 35. The method of claim 34, comprising harvesting before thestep of extracting.
 36. The method of claim 35, comprising cultivatingbefore the step of harvesting.
 37. The method of claim 34, wherein saidpotato starch comprises an amylopectin content of at least 90% and aphosphate content which is increased in comparison to starch fromcorresponding potato plants of the waxy phenotype by at least 30%,wherein said starch from corresponding potato plants of the waxyphenotype is derived from the Desiree variety.